Protoplast-fusion allows efficient, stable transfection of genes carried on plasmid vectors into human cells grown in serum-free media. It provides a new tool for genetic analysis of human cells in vitro, and the means to construct specialized human cells expressing gene products of economic importance. This gene-transfer method originated as a means to fuse dissimilar mammalian cells and thus determine the chromosomal location of genes by following the progeny karyotype during subsequent segreation of chromsomes under conditions which were selective for retention of the gene being mapped. The application of fusion methods to the transfer of bacterial plasmid DNA to mammalian cell recipients has previously emphasized the utility of this method for the study of transient expression of genes as compared to the isolation of genetically stable recombinants as a means to permanently alter the genetic characteristics of human recipient cell populations by recombination. This second application of fusion requires the use of mammalian cells that grow well only in media with high levels of serum and limits its applicability to cell types that are much less sensitive both to toxic contaminants commonly found in reagent grade polyethylene glycol (PETG) and to the manipulations of cell membranes required for fusion transfection. In addition, recombinant episomal DNA isolated from vHa-ras transformed NHBE cells contain Alu-positive human sequences that may be required for replication in human cells.